Type 2 diabetes mellitus (T2DM) ensues from diminished insulin sensitivity and abated compensatory insulin secretion. While diminished insulin secretion has a strong genetic origin, environmental factors are central in the development of insulin resistance; these include hyperinsulinemia and lipotoxicity. Insulin resistance results in the dysregulation of hypothalamic neurons that mediate its central actions: a key intermediary neuron is the neuropeptide Y/agouti-related peptide (NPY/AgRP) neuron. The hypothesis therefore was generated that insulin directly regulates NPY/AgRP neurons, and that prolonged insulin or palmitate, a prevalent free fatty acid (FFA), exposure inhibits neuronal insulin signaling. Using well characterized hypothalamic cell lines, mHypoE-46 or mHypoE-44, which express NPY, AgRP and insulin receptor signaling machinery, this hypothesis was examined in three studies. Correspondingly, insulin decreased NPY and AgRP mRNA expression in the mHypoE-46 cells, through an extracellular signal-regulated kinase (ERK) dependent mechanism; whereas prolonged exposure of NPY/AgRP cells to insulin or palmitate attenuated insulin signaling, determined by analysis of phosphorylated Akt. Insulin induced insulin receptor substrate-1 (IRS-1) serine 1101 phosphorylation in mHypoE-46 cells, utilizing the mTOR-S6K1 pathway, as the mTOR inhibitor rapamycin prevented IRS-1 serine phosphorylation. Insulin also decreased insulin receptor and IRS-1 protein levels; this was prevented by lysosomal and proteasomal pathway inhibitors, 3-methyladenine and epoxomicin, respectively. Importantly, rapamycin, epoxomicin or 3-methyladenine pre-treatment decreased the attenuation of insulin signaling during long-term insulin exposure. On the other hand, palmitate activated c-Jun N-terminal kinase (JNK), the apoptosis effector caspase 3, and induced endoplasmic reticulum (ER) stress in mHypoE-44 cells: JNK inhibition prevented ER stress. In an attempt to avert the deleterious effects of palmitate, the neuronal cells were treated with the 5`AMP-activated protein kinase (AMPK) activator AICAR, a possible insulin sensitizer. Interestingly, AICAR attenuated JNK and caspase 3 activation, and restored insulin signaling. These findings demonstrate that insulin directly regulates NPY/AgRP neuronal cells, and that insulin and palmitate provoke neuronal insulin resistance through different mechanisms. These findings substantiate the idea that environmental factors known to trigger peripheral insulin resistance may have consequences at the level of the individual hypothalamic neuron, which may ultimately contribute to the resulting pathophysiological states of obesity and T2DM.

Combination therapy has proven to be a popular treatment strategy for tighter diabetes control. Since the preliminary evidence is suggestive of complementary actions of American (AG) and Korean Red Ginseng (KRG) in improving glycemia, this project was designed to investigate the therapeutic potential of AG and KRG in combination. Following a randomized, double-blind, placebo-controlled parallel design in a population with diabetes at two centres, the combined use of AG and KRG for 12 weeks was safe, but did not significantly affect glycemic control, blood lipids or blood pressure. However, there was a trend toward lower glycated hemoglobin by 0.7% (p=0.1) and office systolic blood pressure by 5 mm Hg (p=0.052) compared to placebo. These findings encourage further investigation of the mechanism and roles of AG, KRG and their effective components. They also highlight limitations in ginseng research and the need to impose strict regulations to facilitate its standardization.

Combination therapy has proven to be a popular treatment strategy for tighter diabetes control. Since the preliminary evidence is suggestive of complementary actions of American (AG) and Korean Red Ginseng (KRG) in improving glycemia, this project was designed to investigate the therapeutic potential of AG and KRG in combination. Following a randomized, double-blind, placebo-controlled parallel design in a population with diabetes at two centres, the combined use of AG and KRG for 12 weeks was safe, but did not significantly affect glycemic control, blood lipids or blood pressure. However, there was a trend toward lower glycated hemoglobin by 0.7% (p=0.1) and office systolic blood pressure by 5 mm Hg (p=0.052) compared to placebo. These findings encourage further investigation of the mechanism and roles of AG, KRG and their effective components. They also highlight limitations in ginseng research and the need to impose strict regulations to facilitate its standardization.

Type 2 diabetes mellitus (T2DM) ensues from diminished insulin sensitivity and abated compensatory insulin secretion. While diminished insulin secretion has a strong genetic origin, environmental factors are central in the development of insulin resistance; these include hyperinsulinemia and lipotoxicity. Insulin resistance results in the dysregulation of hypothalamic neurons that mediate its central actions: a key intermediary neuron is the neuropeptide Y/agouti-related peptide (NPY/AgRP) neuron. The hypothesis therefore was generated that insulin directly regulates NPY/AgRP neurons, and that prolonged insulin or palmitate, a prevalent free fatty acid (FFA), exposure inhibits neuronal insulin signaling. Using well characterized hypothalamic cell lines, mHypoE-46 or mHypoE-44, which express NPY, AgRP and insulin receptor signaling machinery, this hypothesis was examined in three studies. Correspondingly, insulin decreased NPY and AgRP mRNA expression in the mHypoE-46 cells, through an extracellular signal-regulated kinase (ERK) dependent mechanism; whereas prolonged exposure of NPY/AgRP cells to insulin or palmitate attenuated insulin signaling, determined by analysis of phosphorylated Akt. Insulin induced insulin receptor substrate-1 (IRS-1) serine 1101 phosphorylation in mHypoE-46 cells, utilizing the mTOR-S6K1 pathway, as the mTOR inhibitor rapamycin prevented IRS-1 serine phosphorylation. Insulin also decreased insulin receptor and IRS-1 protein levels; this was prevented by lysosomal and proteasomal pathway inhibitors, 3-methyladenine and epoxomicin, respectively. Importantly, rapamycin, epoxomicin or 3-methyladenine pre-treatment decreased the attenuation of insulin signaling during long-term insulin exposure. On the other hand, palmitate activated c-Jun N-terminal kinase (JNK), the apoptosis effector caspase 3, and induced endoplasmic reticulum (ER) stress in mHypoE-44 cells: JNK inhibition prevented ER stress. In an attempt to avert the deleterious effects of palmitate, the neuronal cells were treated with the 5`AMP-activated protein kinase (AMPK) activator AICAR, a possible insulin sensitizer. Interestingly, AICAR attenuated JNK and caspase 3 activation, and restored insulin signaling. These findings demonstrate that insulin directly regulates NPY/AgRP neuronal cells, and that insulin and palmitate provoke neuronal insulin resistance through different mechanisms. These findings substantiate the idea that environmental factors known to trigger peripheral insulin resistance may have consequences at the level of the individual hypothalamic neuron, which may ultimately contribute to the resulting pathophysiological states of obesity and T2DM.

Seventeen Cree anti-diabetic herbal medicines and eight Traditional Chinese Medicines have been examined for their potential to cause interactions with drugs, which is considered as a major reason for adverse drug effects. Specifically, the effect of these natural health products was examined on major Phase I drug metabolism enzymes including cytochrome P450, human carboxylesterase-1 and flavin-containing monooxygenases. Several of these natural health products have the potential to cause adverse drug effect through the inhibition of major drug metabolism enzymes. The results indicated that 7 Cree medicines plant extracts inhibited CYP3A4 activity, and 3 of them have been proven to cause potent mechanism-based inactivation of CYP3A4. Seven of eight Traditional Chinese Medicines have been identified as strong CYP3A4 inhibitors; the ethanol extract of Goji has identified as a potent inhibitor for CYP2C9 and 2C19. Goji juice showed universal inhibitory effects on most of the tested enzymes except flavin-containing monooxygenases 3.

Es gibt widersprüchliche Befunde darüber, ob depressive Symptome Risikofaktoren für die Neumanifestation eines Diabetes sind oder ob umgekehrt auch Diabetes ein Risikofaktor für depressive Zustände ist. Daher untersuchen wir die längsschnittlichen wechselseitigen Assoziationen zwischen depressiven Symptomen und Typ-2-Diabetes (T2D) sowie die Auswirkungen des gemeinsamen Auftretens beider Erkrankungen auf die Mortalität anhand einer Stichprobe von Hausarztpatienten im Verlauf eines im Mittel 3,5-jährigen Beobachtungszeitraums. Die depressive Symptomatik wurde anhand des Depression Screening Questionnaire (DSQ) kategorial sowie dimensional betrachtet. Die Einteilung in Patienten mit normalem Nüchternblutzucker (NBZ), erhöhtem NBZ sowie T2D (unbehandelt, medikamentös, mit Insulin/kombiniert behandelt) erfolgte nach Arztangaben beziehungsweise nach Laborbefunden zur Baseline-Untersuchung. Die Inzidenz des T2D bei Patienten mit beziehungsweise ohne depressive Symptome betrug 25,6 und 20,9 pro 1000 Personenjahre. Bei dimensionaler Betrachtung des DSQ erhöhte sich das T2D-Risiko (unadjustiert) um das 1,03-Fache [KI (95%): 1,01–1,06] bei punktweisem Anstieg des DSQ. Die Inzidenz depressiver Symptome per 1000 Personenjahre betrug 30,5 für Patienten mit normalem, 34,2 für Patienten mit erhöhtem NBZ, 36,4 für unbehandelte, 32,3 für oral behandelte und 47,8 für insulinbehandelte T2D-Patienten. Verglichen mit Patienten mit einem normalen NBZ hatten insulinbehandelte Patienten ein höheres Risiko für depressive Symptome [HR: 1,71; KI (95%): 1,03–2,83] und oral behandelte T2D-Patienten ein niedrigeres Risiko [HR: 0,58; KI (95%): 0,36–0,96]. Verglichen mit Patienten ohne T2D und depressiver Symptomatik ist das Vorliegen beider Erkrankungen mit einer erhöhten Mortalität assoziiert [HR: 2,49; KI (95%):1,45–4,28]. Die Ergebnisse deuten an, dass vor allem eine Insulinbehandlung bei T2D-Patienten mit inzidenten depressiven Symptomen assoziiert ist. / It is unclear whether depressive symptoms are a risk factor for incident diabetes or diabetes is a risk factor for depressive conditions. Therefore, we examined the longitudinal bidirectional associations between depressive symptoms and type 2 diabetes (T2D) as well as the impact of both diseases on (all cause) mortality in a sample of primary care patients over a 3.5-years follow-up period on average. Depressive symptomatology, defined by the Depression Screening Questionnaire (DSQ), was examined both categorically and dimensionally. Patients were categorized as normal fasting glucose (NFG), impaired fasting glucose (IFG), and T2D (untreated, oral antidiabetics, insulin/combined treatment) according to physician ratings and baseline lab values. Incidence rates of T2D were 25.6 and 20.9 per 1000 person–years for those with and without depressive symptoms, respectively. The unadjusted risk of incident type 2 diabetes was 1.03 times higher (CI(95%): 1.01–1.06) for each 1-point increment in DSQ score. The incidence rates of elevated depressive symptoms per 1000 person–years were 30.5 for NFG, 34.2 for IFG, 36.4 for untreated T2D, 32.3 for oral treated T2D, and 47.8 for insulin/combined-treated T2D patients. Compared to NFG patients, insulin-treated patients had a higher risk of incident depressive symptoms (HR: 1.71; CI(95%): 1.03–2.83) and oral-treated patients had a lower risk (HR: 0.58; CI(95%): 0.36–0.96). Higher mortality rates were associated with both diseases compared to patients without T2D or depressive symptoms at baseline (HR: 2.49; CI(95%):1.45–4.28). Results indicate that especially insulin treatment in T2D patients is associated with incident depressive symptoms.

To gain insight in the relationship between innate immune system and metabolic disease, we aimed to investigate the effects of lactoferrin in obesity-related metabolic disturbances. Circulating lactoferrin concentration was significantly decreased in subjects with altered glucose tolerance (AGT) and associated negatively with obesity-related metabolic disturbances. The SNPs-induced aminoacidic changes in lactoferrin N-terminus region were associated with a low atherogenic lipid profile. Lactoferrin production in neutrophils decreased significatively in aging, chronic low-grade inflammation and type 2 diabetes. In vitro, lactoferrin increased insulin signaling pathway, even under insulin resistance conditions and displayed dual effects on adipogenesis (antiadipogenic in 3T3-L1 and adipogenic in human adipocytes). In conclusion, lactoferrin might play a potential protective role against insulin resistance and obesity related metabolic disturbances. / Per aprofundir en la relació entre el sistema immunològic innat i els trastorns metabólics, s’investiga l’efecte de la lactoferrina en els desordres metabòlics associats a l’obesitat. Els nivells circulants de lactoferrina es trobaven significativament disminuits en subjetes amb la tolerància a la glucosa alterada (AGT), i aquests es correlacionaven negativament amb trastorns metabòlics associats a obesitat i resistència a la insulina. Els canvis aminoacídics en la seva regió N-Terminal s’associaven a un perfil lipídic menys aterogènic. La producció de lactoferrina es troba reduïda en condicions d’envelliment, inflamació crònica i diabetes tipus 2. In vitro, la lactoferrina incrementava la via de senyalització de la insulina, inclús en condicions de resistència a la insulina i presentava efectes dual en la adipogenesis (antiadipogenic en 3T3-L1 i adipogenic en adipòcits humans). En conclusió, la lactoferrina podria tenir un potencial efecte protector enfront les enfermetats metabòliques associades a obesitat i resistència a la insulina.

Adipositas ist besonders in den Industrienationen ein zunehmendes gesellschaftliches und ökonomisches Problem. Dabei sind vor allem die kardiovaskulären und metabolischen Begleiterkrankungen von entscheidender Bedeutung. In den letzten Jahren konnte gezeigt werden, dass verschiedene Adipozyten- und Hepatozyten-sezernierte Proteine Mediatoren von Insulinresistenz und Dyslipidämie darstellen. Kürzlich wurde Angiopoietin-related growth factor (AGF) als ein neues, von der Leber produziertes Protein, das potentiell Insulinresistenz und Adipositas antagonisiert, vorgestellt. Im Mausmodell waren AGF-überexprimierende Tiere schlanker und insulinsensitiver verglichen zu Kontrolltieren. Zudem entwickelten AGF-knockout-Mäuse eine Adipositas, Insulinresistenz sowie eine Leber- und Skelettmuskelverfettung. Weiterhin fand sich in epidermalen Keratinozyten eine Hypervaskularisierung bei transgenen Mäusen mit AGF-Expression. Dies macht AGF möglicherweise zu einem Zielgen in der Behandlung moderner Zivilisationskrankheiten, wie z.B. dem Diabetes mellitus Typ 2 (DMT2). Bisherige Publikationen über AGF basieren zumeist auf Tiermodellen. Über die Regulation beim Menschen existieren dagegen bislang nur wenige Studien. In der vorliegenden Arbeit wurde AGF im Serum verschiedener Patientenpopulationen mit einem erhöhten kardiovaskulären Risikoprofil (Patienten mit chronischer Dialysepflicht, DMT2, Präeklampsie [PE]) mittels enzyme-linked immunosorbent assay quantifiziert und mit Kontrollpatienten verglichen. Die Ergebnisse zeigen, dass AGF bei Patienten mit DMT2 im Vergleich zu Nichtdiabetikern signifikant erhöht ist. Bei terminal-niereninsuffizienten Patienten dagegen fanden sich signifikant niedrigere AGF-Konzentrationen im Serum. Bei PE-Patientinnen waren signifikant höhere AGF-Spiegel nachweisbar verglichen zu gesunden schwangeren Kontrollen. Die vorgestellten Daten weisen darauf hin, dass erhöhte AGF-Spiegel bei DMT2 und PE eine physiologische Gegenregulation darstellen könnten, die der Insulinresistenz bei DMT2 bzw. antiangiogenetischen Faktoren bei PE entgegenwirkt. Alternativ wäre – ähnlich der Insulinresistenz – eine Resistenz von Patienten mit DMT2 bzw. PE gegen AGF möglich mit einer reflektorischen Erhöhung dieses Hepatozyten-exprimierten Faktors. Die genaue Rolle von AGF bei kardiovaskulären Risikopatienten muss in zukünftigen Arbeiten noch weiter aufgeklärt werden.

Type 2 diabetes is a global disease which affects an increasing number of peopleevery year. At the heart of the disease lies insulin resistance in the target tissues,primarily fat and muscle. The insulin resistance is caused by the failure of a complexsignalling network, and several mechanistic hypotheses for this failure havebeen proposed. Herein, we evaluate a hypothesis that revolves around the proteinmammalian target of rapamycin (mTOR) and its feedback signals to insulin receptorsubstrate-1 (IRS1). In particular, we have re-examined this hypothesis andrelevant biological data using a mathematical modelling approach. During the course of modelling we gained several important insights. For instance,the model was unable to reproduce the relation between the EC50-valuesin the dose-response curves for IRS1 and its serine residue 312 (Ser-312). Thisimplies that the presented hypothesis, where the phosphorylation of Ser-312 liesdownstream of the tyrosine phosphorylation of IRS1, is inconsistent with the provideddata, and that the hypothesis or the data might be incorrect. Similarly, wealso realized that in order to fully account for the information in the dose-responsedata, time curves needed to be incorporated into the model. A preliminary model is presented, which explains most of the data-sets, butstill is unable to describe all the details in the data. The originally proposed hypothesisas an explanation to the given data has been revised, and our analysisserves to exemplify that an evaluation of a mechanistic hypothesis by mere biochemicalreasoning often misses out on important details, and/or leads to incorrectconclusions. A model-based approach, on the other hand, can efficiently pin-pointsuch weaknesses, and if combined with a comprehensive understanding of biologicalvariation and generation of experimental data, mathematical modelling canprove to be a method of great potential in the search for mechanistic explanationsto the cause of insulin resistance in type 2 diabetics.

Observational studies have indicated that fruit and vegetables, and dietary antioxidants may play an important role in reducing the risk of chronic diseases, potentially by affecting pathogenic mechanisms such as oxidative stress and inflammation. Clinical trials investigating the effects of supplementation with single or a few antioxidants in high doses have, however, shown inconsistent results and thus have not been able to support the observational findings. It was therefore hypothesised that a supplement, containing a combination of antioxidants mainly extracted from fruit and vegetables, and supplied at moderate doses, might act more beneficially than single antioxidants given at pharmacological doses. The effects of such a supplement were investigated in two interventional studies described in this thesis. The effects on antioxidant status, metabolic control, oxidative stress and inflammation were investigated in overweight men and in patients with type 2 diabetes, subjects that could be expected to have elevated levels of oxidative stress and inflammatory activity. The results of the studies did not support the hypothesis that supplementation with antioxidants from fruit and vegetables may have beneficial effects by counteracting oxidative stress and inflammation, despite markedly increased plasma antioxidant concentrations. However, interesting associations were observed in diabetes patients at baseline between intake of antioxidant rich food as well as levels of antioxidants in plasma, and markers of oxidative stress and inflammation. These associations are compatible with the hypothesis that a high intake of fruit and vegetables and dietary antioxidants decrease oxidative stress levels, have anti-inflammatory effects and a beneficial influence on glycaemic control. The results also indicated that glycaemic control may affect the level of oxidative stress. The absence of beneficial effects from antioxidants might to some extent be explained by the initial levels of oxidative stress and inflammation and by the antioxidative status in the subjects included in the studies. Since the levels generally were comparable with those observed in healthy subjects, this might have decreased the ability to observe any beneficial effects of supplementation with additional antioxidants. Continued investigations are needed to characterise the individuals who potentially might benefit from antioxidant supplementation. In view of apparent positive effects from a high intake of fruit and vegetables found in observational studies and until more knowledge is available from interventional trials about possible benefits and potential risks of antioxidant supplementation it still seems reasonable to recommend a diet rich in fruit and vegetables.